Method of fermenting a mycocidal antibiotic of polyene series

ABSTRACT

A process for producing the antifungal antibiotic by a fermentation levorin process consisting in cultivation of Actinomyces levoris jointly with yeast or yeast-like fungi of the genera Candida, Saccharomyces or Monilia on a culture medium containing sources of carbon and nitrogen and mineral salts, e.g., maize meal, soya meal, hydrol and sodium chloride, until a maximum accumulation of the antibiotic is obtained therein.

United States Patent [191 Tsyganov et al.

Inventors: Vladimir Alexeevich Tsyganov,

Vladimirskaya, 38, kv. 21, Leningrad, all of USSR.

prospekt Geroev, 20, kv. 8, Leningrad; Valentina Pavlovna Namestinikova, l4 Linia V.O. 31/33, kv. 86, Leningrad; Valentina Vasilievna Antonova, prospekt Engelsa, 22, kv. 123, Leningrad; Esfir Iliinichna lofina, ulitsa Volodarskogo, 69, kv. 46, Penza; Mark Abovich Malkov, ulitsa Bogdanova, 6, kv. 55, Penza; Vladimir Semenovich Nyn, ulitsa Druzhby, 5, kv. 1 1, Penza; Olga Alexandrovna Kuznetsova, Uglovoi pereulok, 6, kv. 10, Leningrad; Galina Nikolaevna Malinovskaya, naberezhnaya Chernoi rechki, 51, kv. 38, Leningrad; Galina Petrovna Poluparneva, ulitsa Vosstania, 1, kv. l8, Leningrad; Anastasia Mikhailovna Anosova, naberezhnaya reki Fontanki, 166, kv. 31, Leningrad; Tamara Kirillovna Pudakova, ulitsa Druzhby, 3, kv. 17, Penza; Raisa Ivanovna Judina, ulitsa Druzhby, 5, kv. 5, Penza; Elena Pavlovna Yakovleva, ulitsa Druzhby, 3, kv. 20, Penza; Vladimir Mikhailovich Morozov, ulitsa [22] Filed: Feb. 25, 1971 [21] Appl. No.: 119,014

[52] US. Cl. 195/80 R, 195/96 [51] Int. Cl Cl2d 9/00 [58] Field of Search 195/80 R, 111,96

[56] References Cited OTHER PUBLICATIONS Chem. Abstracts, Volume 66, 1967, 5748n.

Primary Examiner-A. Louis Monacell Assistant E.\-aminer-Robert J. Warden Attorney, Agent, or Firm-Waters, Roditi, Schwartz & Nissen 5 7] ABSTRACT A process for producing the antifungal antibiotic by a fermentation levorin process consisting in cultivation of Actinomyces lei oris jointly iaiith yeast" bi' ea'srlike fungi of the genera Candida, Saccharomyces or Monilia on a culture medium containing sources of carbon and nitrogen andmineral salts, e.g., maize meal, soya meal, hydrol and sodium chloride, until a maximum accumulation of the antibiotic is obtained therein.

4 Claims, N0 Drawings METHOD OF FERMENTING A MYCOCIDAL ANTIBIOTIC OF POLYENE SERIES The present invention relates to a process for producing the antifungal antibiotic levorin by a fermentation process comprising the cultivation of Actinomyces levoris (deposited under N0. 879 at the Culture Museum of the All-Union Research Institute of Antibiotics, Moscow, USSR) jointly with yeasts or yeast-like fungi of the genera Candida, Saccharomyces, Monilia.

Actinomyces levoris is characterized by the following taxonomic description.

Spore-carriers non-spiral, straight, short slightly wavy sometimes, elongated, but having no spiral coils, arranged one by one, seldom in clusters, Spores ovalshaped, elongated, with a smooth envelope.

The cultures are well grown on synthetic or organic culture media. The colonies are colorless, the media do not become colored. Air mycelium is pale-yellow, white-pale-yellow or yellow-cream in color. In appearance they look similar to the cultures Act. streptomycini.

CPI-Chapek-agar-moderate growth, colonies colorless, culture media do not become colored. Air mycelium is white-pale yellow, fluffy or flour-like.

CPII good growth, colonies colorless or brightyellowish from underside, the medium uncolored. Air mycelium white-pale-yellow, fluffy or flour-like.

Glucose asparaginic agar-good growth, colonies and culture medium uncolored. Air mycelium pale-yellow.

Potato agar abundant growth, colonies colorless or s]ightly-yellowish-brownish, culture medium uncolored. Air mycelium white or pale-yellow.

Fish agar-good growth, colonies and culture medium colorless; sometimes slightly yellowish from underside when the colony gets older. Air mycelium dark straw-colored.

MIIA moderate growth, colonies and culture medium colorless. Air mycelium absent or scarce, whitish.

Ml'lB good growth, when agitating the cultures in shaking flasks air mycelium disintegrated into stick-like elements.

Potato slices, colonies dry, crust-like, with a scarce layer of whitish mycelium, culture medium gets slightly brown.

Gelatine is readily diluted, milk is peptonized after coagulation, starch intensely hydrolyzed, saccharose inverted.

Tyrosinase negative, no melanoid formation, no gro-. wth on cellular tissue, nitrates reduced to nitrites, Tresner-Dang medium does not become black.

Glucose, maltose, galactose, arabinose, glycerine, mannite and starch assimilated.

Saccharose, lactose, raff'mose, ramnose, inosite, inuline not assimilated.

The present species is characterized by active suppression of yeast-like organisms. All of the strains strongly suppress the growth of sporogenic and asporogenie yeast from the groups of Torula, Pichia, Saccharomyces, Willia, Candida, Monilia, etc. They have the same activity in suppressing the growth of many fungi:

Licillium dahliae, Bottytis allii, Deuterophoma tracheiphila, Aspergillus niger, Penicillium chrysogenum, Alternana humicala, Phomopsis cinerea. Exert' no action upon bacteria or suppress one or two species of gram-' positive bacteria, mainly Bac.id0sus and Mycob. luteum. Do not suppress the growth of gram-negative bacteria.

In accordance with the recent investigations, our strains are the most similar to Act. candicidinus producer of an antibiotic candicidine and Act. canescus producer of askasine.

Levorin appertains to heptaene macrolide antibiotics and finds application in medical practice for treatment of candidiases of the gastrointestinal tract and internal organs, and can be applied locally for treatment of candidiases of the skin, mucous membranes and genitals.

Known heretofore are methods of producing the antibiotic levorin consisting in that the producer of levorin, viz.', a ray fungus Actinomyces levoris is grown as a monoculture on a variety of culture media or fermentation broths containing various mineral and organic substances (in the form of a solution or a water suspension) necessary for the producer to grow and form the antibiotic, such as glucose, maize meal, sodium chloride and calcium carbonate.

Then the culture medium is sterilized, whereupon a suspension of the antibiotic producer is inoculated thereinto and said producer is grown until a maximum accumulation of the antibiotic is obtained therein.

The resulting culture medium has an activity of 15,000-20,000 units/ml (cf. USSR Autho'rs Certificate No. 187244; Antibiotiki, 1969, vol. 4, No. 4, pp. 21-25.

However, the above-discussed methods suffer from a disadvantage that the resultant culture medium possesses but low activity. It is therefore a primary object of the present invention to eliminate said disadvantage.

According to the above-said general object the invention has a specific object to enhance the activity of the culture medium obtained by enrichment of said medium in the process of cultivating Actinomyces levoris.

Said object is achieved due to the fact that in a process for producing the antifungal antibiotic, levorin by growing Actinomyces levoris on a culture medium containing sources of carbon, and nitrogen, as well as mineral salts until maximum antifungal activity is imparted to said culture medium, according to the invention the culture of Actinomyces levoris is grown on the culture medium concurrently with yeasts or yeast-like fungi of the Candida, Saccharomyces and Monilia genera. To enhance the activity of the resultant culture medium the growth process preferably takes place on a culture medium of the following weight percent composition:

maize meal 2,

soya meal l,

hydrol (with respect to reducing agents) 1,

sodium chloride 0.5,

calcium carbonate ().l,

water constituting the remainder. The culture of yeasts or yeast-likejung is inoculated into the culture medium in an amount of 1,'.4 vol. percent.

The fermentation process can be carried out by growing yeasts or yeast-like fungi on the culture medium for 24-48 hours followed by inoculating with the culture of Actinomyces levoris or by simultaneously growing on the same culture medium the culture of yeasts or yeast-like fungi and the culture of Actinomyces levoris.

The method disclosed herein is carried into effect as foliows:

To prepare the inoculum the culture of Actinomyces levoris is grown on an agar medium of the following composition: dipotassium hydrogen phosphate 1 g; sodium nitrate 3 g; potassium chloride 0.5 g; magnesium sulfate 0.5; ferric sulfate 0.015 g; glucose 20 g; agar .-25 g; per liter of water, the pH value being 7.0-7.2. The growth process runs at 2728C for 10 days; thereupon the inoculation material is placed into 750 ml. shaking flasks rotating at 220 rpm, each flask containing culture medium of the following composition:

soya meal 10 g; glucose 10 g; sodium chloride 5 g; calcium carbonate l g; per liter of water. Growing in the flasks is conducted at 2728C for 48 hours.

For a joint cultivation of Actinomyces Levoris and yeasts or yeast-like fungi a culture medium containing sources of nitrogen and carbon, and mineral salts is used. It is expedient to use a mixed soya-maize-hydrol culture medium of the following composition: maize meal 20 g; soya meal g; hydrol 10 g; sodium chloride 5 g; calcium carbonate 1 g; per liter of water.

The above-said culture medium is dispensed to 750 ml shaking flasks in 50 ml amounts and then is sterilrzeaarizo cirar 3 0515.

For a joint cultivating .the following species of yeasts and yeast-like fungi are suitable for use: Candida albicans, Candida Krusei, Candida tropicalis, Candida pseudotropicalis, Saccharomyces cerevisia, Monilia m'vea, Candida parapsilasis and others.

Yeasts or yeast-like fungi are grown beforehand on agar wort at 37C for 24 hours. In case of joint cultivation 1-4 vol. percent of the yeasts suspension are introduced into the flasks containing the culture medium. Then the flasks are placed on a shaker and incubated for 24-28 hours at 2728C, whereupon 10 vol. percent of the Acrinomyces Levoris inoculum grown as described above are inoculated into the flasks. The fermentation process is carried out for 72-120 hours at 2728C.

The activity of the resulting culture liquid is found to amount to 65,000 units/ml.

Another way of carrying out a concurrent cultivation of yeasts and yeast-like fungi with Actinomyces Levoris is accomplished by simultaneous inoculation of Actinomyces levoris and yeasts or yeast fungi into the culture medium. To this end, 2-4 vol. percent of the yeast suspension are introduced into shaking flasks containing a mixed soya-maize-hydrol culture medium of the abovespecifled composition, where upon the flasks are placed on a shaker and incubated for 24-48 hours. At the same time a 48 hr inoculum of Actinomyces Ievoris is prepared. Then the yeast inoculum and the inoculum of Actinomyces Levaris are simultaneously introduced into the culture medium, and fermentation is carried out at 27-'-28C for 72-120 hours. The activity of the resultant culture liquid amounts to 55,000 units/ml. v

The herein-proposed method ensures an enrichment of the culture medium with proteins, carbohydrates, vitamins and other growth factors by introducing yeasts and yeast-like fungi thereinto, and due to this increases the level of accumulation of antibiotic activity by 50 percent as compared to the methods used heretofore.

Furthermore, the application of the culture medium of the herein-disclosed composition, containing soya meal and hydrol, makes it possible to render the levorin content in the culture liquid as high as 2.8 times as compared with conventional culture media.

in what follows the nature of the present invention will be disclosed by examples of carrying out the process for producing the antifungal antibiotic levorin.

EXAMPLE 1 2 ml of a 10 cell suspension of Saccharomyces cerevisiae were introduced into 750 ml. shaking flasks rotating at 220 rpm, each flask containing 50 ml of a mixed soya-maize-hydrol culture medium of the following composition: maize meal 20 g, soya meal 10 g, bydrol 10 g, sodium chloride 5 g, calcium carbonate 1 g per liter of water.

To obtain a 10 cell suspension of the above-said culture, the latter was grown on agar wort at 37C for 24 hours and then washed off with physiological saline solution.

After introduction of said suspension the culture was grown at 27C for 24 hours.

Then 5 ml. of a 48 hr. inoculum of Actinomyces levoris were introduced under sterile conditions into the flasks containing the prepared yeast mass, said inoculum being obtained by growing on a culture medium of the following composition: soya meal 10 g; glucose 10 g, sodium chloride 5 g, calcium carbonate l g per liter of water, at 2728C for 48 hours. Then fermentation was carried out at 27C for 6 hrs. The culture liquid resulting from a joint growing of the yeasts Saccharomyces cerevisiae and Ac tinomyces Levoris was found to possess an activity of 62,300 units/ml.

EXAMPLE 2 1 m1 of a 10 cell suspension of Candida tropicalis was introduced into 750 mi. shaking flasks rotating at 220 rpm, each flask containing 50 ml of a mixed soyamaize-hydrol culture medium of the following composition: maize meal 20 g, soya meal -l0 g, hydrol 10 g, sodium chloride 5 g, calcium carbonate l g per liter of water.

To obtain a 10 cell suspension of the above-said culture, the latter was grown on agar wort at 37C for 24 hours and then washed off with physiological saline solution. After introduction of the aforesaid suspension, the culture was at 27C for 48 hours. Then into the flasks with the prepared yeast mass were introduced under sterile conditions 5 ml of a 48hr inoculum of Actinomyces levoris, obtained in a manner similar to that described in Example 1. Then fermentation was carried out at 2728C for hours.

The culture liquid resulting from a concurrent growing of Candida tropicalis and Actinomyces Levoris had an antibiotic activity of 64,100 units/ml.

EXAMPLE 3 2 ml of a 10 cell suspension of Monilia nivea were introduced into 750 ml. shaking flasks rotating at 220 rpm, each flask containing 50 ml of a culture medium of the composition similar to that in Example 1 To obtain a 10 cell suspension of the above-said culture, the

latter was grown on agar wort at 37C for 24 hours and then washed off with physiological saline solution. After introduction of said suspension, the culture was grown at 27C for 24 hours. Then 5 ml ofa 48 hr inoculum of Actinomyces levoris were introduced into the flasks containing the prepared yeast mass, said inoculum being obtained in the same way as described in Example l. The fermentation process was carried out at 2728C for 72 hours. The culture liquid resulting from a concurrent growing of Monilia nivea and Actinomyces Levoris had an activity of 61,800 units/ml.

EXAMPLE 4 4 ml of a cell suspension of Candida tropicalis were introduced into 750 ml. shaking flasks at 220 rpm, each flask containing 50 ml of a culture medium of the following composition glucose g, soya meal 20 g, calcium carbonate 1 g per liter of water, the pH being 6.8-7.2. To obtain a 10 cell suspension of the above-said culture, the latter was grown on agar wort at 37C for 24 hours and then washed off with physiological saline solution. After introduction of said suspension, the culture was grown at 27C for 48 hours. Then 5 ml of a 48 hr. inoculum of Actinomyces levoris were introduced, under sterile conditions, into the flasks containing the prepared yeast mass, said inoculum being obtained in the same way as described in Example l. Then fermentation was carried out at 2728C for 120 hours to obtain the culture liquid with an activity of 31,700 units/ml.

EXAMPLE 5 2 ml of a 10 cell suspension of Candida tropicalis were introduced into 750 ml. shaking flasks rotating at 220 rpm, each flask containing 50 ml of a mixed soyamaize-hydrol culture medium of the following composition: maize meal 20 g, soya meal 10 g, hydrol 10 g, sodium chloride 5 g, calcium carbonate-- 1 g per liter of water. To obtain a 10 cell suspension of the above-said culture, the latter was grown on agar wort at 37C for 24 hours and washed off with physiological saline solution. The culture introduced into the flask was grown at 27C for 48 hours.

Then 5 ml of the resulting yeast culture of Candida tropicalis and 5 ml of the inoculum of Actinomyces levoris, obtained in the same way as described in Example 1, were introduced into 250ml. shaking flasks, each containing 50 ml of the above-said mixed soyamaize-hydrol culture medium, whereupon fermentation was carried out at 27C for hours. The culture liquid resulting from a joint growing of the yeasts Candida tropicalis and Actinomyces Levoris was found to possess an activity of 53,700 units/ml.

What is claimed is:

1. A process for producing the antifungal antibiotic levorin comprising cultivating a culture of Actinomyce's levoris jointly with yeasts or yeast-like fungi selected from the group consisting of the genera Candida, Saccharomyces and Monilia on a culture medium containing sources of carbon and nitrogen and mineral salts, until maximum antifungal activity is imparted to said culture medium.

2. A process as claimed in claim -1 wherein the growth process is carried out on a culture medium of the following weight percent composition: maize meal 2, soya meal l, hydrol (with respect to reducing agents) 1, sodium chloride 0.5, calcium carbonate 0.1, water constituting the remainder.

3. A process as claimed in claim 1, wherein the culture of yeasts or of yeast-like fungi is introduced into the culture medium in an amount of 1-4 vol. percent.

4. A process as claimed in claim 1 wherein the yeasts or yeast-like fungi are grown on a culture medium for 24 48 hours, whereupon the culture of Actinomyces levoris is inoculated thereinto. 

2. A process as claimed in claim 1 wherein the growth process is carried out on a culture medium of the following weight percent composition: maize meal - 2, soya meal - 1, hydrol (with respect to reducing agents) - 1, sodium chloride - 0.5, calcium carbonate - 0.1, water constituting the remainder.
 3. A process as claimed in claim 1, wherein the culture of yeasts or of yeast-like fungi is introduced into the culture medium in an amount of 1-4 vol. percent.
 4. A process as claimed in claim 1 wherein the yeasts or yeast-like fungi are grown on a culture medium for 24 - 48 hours, whereupon the culture of Actinomyces levoris is inoculated thereinto. 